One of the grand open challenges in modern science is to identify cells or probe molecules and understand the mechanism and dynamics of biological processes at the molecular level with high spatiotemporal resolution, and particularly inside living cells and tissue. As a result of the wealth of information potentially accessible from such biological targets, there has been a growing demand for imaging tools for biomedical research and medicine. This research has led to the development of new techniques like magnetic resonance imaging (MRI), ultrasound, positron emission tomography (PET), and optical coherence tomography (OCT). However, these techniques require high costs and some fundamental technological barriers hinder their widespread use.
Optical imaging is a practice that has recently gained widespread clinically relevant use that utilizes photons as an information source to analyze cells and tissues at multiple length and time scales, with applications in a wide range of basic science and clinical studies like pharmacology, cellular biology, and diagnostics. For example, semiconductor nanocrystals, small organic dyes or fluorescent proteins are commonly used as optical labels in in vivo optical imaging. (See, e.g., X. Michalet et al., Science 307, 538 (Jan. 28, 2005); B. Dubertret et al., Science 298, 1759 (Nov. 29, 2002); M. K. So, C. Xu, A. M. Loening, S. S. Gambhir, J. Rao, Nat Biotechnol 24, 339 (March, 2006); N. C. Shaner, P. A. Steinbach, R. Y. Tsien, Nat Methods 2, 905 (December, 2005); and B. N. Giepmans, S. R. Adams, M. H. Ellisman, R. Y. Tsien, Science 312, 217 (Apr. 14, 2006), the disclosures of which are incorporated herein by reference.) Indeed, recent advances in fluorescence microscopy alone have profoundly changed how cell and molecular biology is studied in almost every aspect. (For example, see, Lichtman, J. W. & Conchello, J. A. Nat. Methods 2, 910-919 (2005); Michalet, X. et al. Annu. Rev. Biophys. Biomolec. Struct. 32, 161-182 (2003); Jares-Erijman, E. A. & Jovin, T. M. Nat. Biotechnol. 21, 1387-1395 (2003); Bastiaens, P. I. H. & Squire, A., Trends Cell Biol. 9, 48-52 (1999); and Suhling, K., et al, Photochem. Photobiol. Sci. 4, 13-22 (2005), the disclosures of which are incorporated herein by reference.)
However, the ultimate need of characterizing biological targets is largely unmet due to fundamental deficiencies associated with the use of fluorescent agents. For example, fluorescent probes face two major limitations that have a significant impact on the signal strength: 1) dye saturation, because the number of photons emitted by the fluorophore in a given time is restricted by the excited state lifetime, and 2) dye bleaching, which limits the total number of photons produced per dye. In addition, autofluorescence from tissue organic components after illumination absorption can severely limit the signal-to-noise ratio of fluorescence imaging experiments. Finally, fluorescence is fundamentally an optically incoherent process, and as a result extracting 3D information from the source is inherently difficult.
To overcome these limitations, a new kind of second harmonic generating (SHG) imaging nanoprobe has been developed. These SHG nanoprobes are characterized by photophysical properties that are fundamentally different to conventional probes, such as fluorescent agents. In particular the nonlinear nanocrystal SHG nanoprobes such as barium titanate (BaTlO3) provide a unique combination of advantageous properties inherent to the SHG process that allow experiments characterizing molecular targets with excellent sensitivity for an indefinite length, with fast acquisition rates and superb signal-to-noise ratio (SNR). Accordingly, it has been recognized that SHG nanoprobes offer great potential to give insights into the dynamics of various biological targets at the molecular level with unmatched sensitivity and temporal resolution for both molecular imaging and clinical diagnostics. (See, e.g., U.S. Pat. Pub. Nos. 2012-0141981 and 2010-0233820, the disclosures of which are incorporated herein by reference.)
However, because inherent material properties targeting mechanism do not provide the targeting or delivery characteristics desired, methods to modify SHG nanoprobes with relevant chemical and biological agents are needed. Accordingly, a need exists for functionalized and targeted SHG nanoprobes, and methods of functionalizing and targeting these nanoprobes.